Coconut milk extracts



P 1957 F. c. STEWARD ET AL 2,806,027

COCONUT MILK EXTRACTS Filed July 13, 1955 2 Sheets-Sheet 1 2000 l I I I v I600- I00 2 80 I200- A a 60 g 2 800 z 40 E 2-, 400

Q 1 1 l 1 I I I I l 0 l l I 2-0 4.0 6.0 8.0 [0.0 [2.0 14.0 200 20 300 WAVELENGTH lN MICRONS WAVELENGTH IN mp 0 I l l I 20 40 60 80 300 20020 40 so so 300 20 WAvELENGTHIN'mu WAVELENGTH IN mp INVENTORS FREDERICK c. STEWA RD EDGAR M. SHANTZ BY (TM 564%,

ATTORNEY Sept. 10, 1957 F. c. STEWARD ETAL 2,806,027

COCONUT MILK EXTRACTS Filed July 13, 1955 2 Sheets-Sheet 2 /0 TRANSMISSION l l l l l 1 l 1 l l Z0 4.0 6.0 8.0 l0-0 I I4.() I61) WAVELENGTH lN MICRONS GROWTH ASSAY OF COMPOUND A Basal medium onl Basal coconut ml Basal casein hy drolysale Basal compound A l.O p.p.m Basal casein hydr. compd. A' l.O p.p.m.

GROWTH ASSAY OF COM POUND"B" Basal medium onl Basal +coconul m|l Basal casein hydrolysate Basal compd-B' 0.25 p.p.m. Basal casein hydn+ compd.'B 0.25 p.p.m.

GROWTH ASSAY OF COMPOUND"C" Basal medium onl Basal coconul mil Basal casein hydrolysale Basal compound'C' 2.0 p.p.m. Basal casein hydr. compd'C' 2.0 ppm.

l l l l l l I Q FlNAL FRESH WElGHT lN MlLLlGRAMS INVENTORS FREQERICK C. STEWARD EDGAR M. SHANTZ ATTORNEY United States atent has! COCONUT MILK EXTRACTS Frederick C. Steward and Edgar M. Shantz, Ithaca, N. Y. Application July 13, 1955, Serial No. 521,860 Claims. (Cl. 260-2365) This invention relates to a process for obtaining crystalline substances capable of inciting in the presence of a basal medium and casein hydrolysate random cell division in plant tissues, and to the substances so obtained.

This application is a continuation-impart of our copending application Serial No. 365,182, filed June 30, 1953 now abandoned.

More particularly, the invention centration process comprising the steps of diluting whole coconut milk with alcohol, treating the diluted milk with mercuric acetate, filtering the resulting precipitate, suspending the precipitate in water, treating the suspended precipitate with hydrogen sulfide, filtering oi? the precipitated sulfide, collecting and concentrating the filtrate to a heavy sludge and extracting the sludge with alcohol to yield an extract of growth-promoting substances.

When whole coconut milk is added to a basal medium containing mineral salts, sugars and vitamins it causes a striking increase in the growth by cell division of explants of certain tissues, notably carrot root phloem. Whole coconut milk produces an optimum growth response when added to the culture medium at a level of about 15% by 700 gallons coconut milk (active at 10,000 p.

pH 5.0 dry weight is directed to a con I volume, which represents a concentration parts per million on a dry weight basis.

We have now found that by subjecting coconut milk to a series of extractive and fractionation steps in definite sequence recognizable chemical entities are obtained, which in a basal medium containing casein hydrolysate give a plant growth response equivalent to that given by the addition of whole coconut milk. The chemical entities obtained are also active at much lower concentrations.

The term coconut milk as used herein refers to the fluid endosperm that nourishes an immature embryo which later produces a spongy mass of cotyledonary tissue that eventually fills the central cavity of the seed. Coconut milk activity refers to the dramatic ability of this fluid to foster rapid and random division of otherwise mature cells of higher plants.

The initial enrichment of the coconut milk activity is made by reacting whole coconut milk with an excess of mercuric acetate after dilution by an equal volume of a lower aliphatic alcohol, preferably ethanol. After filtration, the resulting precipitate is suspended in water, treated with hydrogen sulfide and filtered to remove the pre cipitated sulfide. The filtrate is concentrated to a heavy sludge under reduced pressure or by any other conventional method.

The sludge is then extracted with a lower alcohol such as for example ethanol. The sludge is agitated during the extraction. The removal of the solvent leaves a dark heavy syrup equivalent to about 0.6% of the initial dry material of the coconut milk.

Such an extract as above described can be fractionated further by differential solubilities in any of a variety of solvents and by partition chromatography on cellulose. The methods by which the isolation of growth-promoting substances in coconut milk has been achieved are shown below in flow sheet form.

of about 10,000

Diluted with equal vol. alcohol, Excess Hg acetate added Eg precipitate Suspended in water treated. with Hi8 Preclpitate Flltrate HgS Flltrate (inactive) Solvent removed under reduced pressure Extracted with 85% ethanol Residue (inactive at 1,000 p. p. m.)

Extract, 4.3 lbs;

(active at -250 p. p. m.)

2,808,027 a p a 3 4 Y V l i! I! Extraction with strongly Ether extract 200 gms aqueous phase filtered acidified ethanol (inactive) 7 Residue Filtrate of ether-extracted Crude C. M. F. conc. No. 2. r r 500 mgms. crystal1ine sulphur alcohol-refined C. M. F. concen- (active at 200-500 p. p. in.) l trate (active at 50-200 p. p. m.)

70 mgms. crystalline material l picked from brown residue 7 on filter paper seenextpage for further-treatment oNi ciiude C. F. concentrate l V o.

Recrystallized twice from hot ethanol Fine white crystalline needles (active at 1-5 p. p. m. on explants from some carrots), but only in conjunction with casein hydrolysate. (Crystalline compound A.)

" Crude M. F: concentrate N o. 1=300 gallons of coconut milk zxalcohol-refinedfiether extracted (active at 50-200 p. p. m.)

'l v l 100'gms. in aqueous'solution Distillation in high-vacuum pot I extracted with n-butanol still. 1 gin. heated '45 min-at 235 0., 5 [is pressure.

\i butanol extract 2.0 grams aqueous phase 93 grams v i F dissolved in ml.- water (slightly active at 50 distillate mgms. (inactive) residue 1.96 gms. (active at 100 p. p. m.) i Dv P- 7 1 Charcoal chromatography 250 gms;'adsorbed' on'l80 g'ms: charcoal (Darco (3-60) V Compound D (mact) water-insoluble ppt. (40 mgms.) water soluble portion 1.9G.gms. 7 197 grams (no absorption at 260 washed with water (active on some carrots at 20 (active at 5 p. p. m.) V 1111;)(11120171V6) p. p. m. but only with added casein hydrolysate) l washed with glacial acetic acid Precipitate from mixtures of I I benzene-alcohol 300 mgms. l z 18.0 grams (strong absorption at \\J 260 m (active at 10-50 p. p. m.)

iractionsolublein90/10benzcne-ethanol88mgms. fraction insoluble in /50 benzene- 45 grams of original weight and of Pale yellow, no u. v. absorption at 260 m (acethanol 99 mgms.darkred,strongu. v. u. v.absorbing material non-recoverable tive at 2-10 p. p. m.) absorpti)on at 260 my (activei at 2-10 from column.

p. p. m.

intermediate fractions 113 extracted with absolute ethanol mg'ms. (active) V l insoluble residue (inactive) soluble portion 7.6 grams (active) recrystallized x 3 from hot water active materials to be worked-upby partition r chromatography l l r i pure tyrosine (inactive) Chromatography on cellulose columns. Moving phase=1z-butanol saturated with 10% aqueous acetic acid.

In general, the activity appears distributed throughout fractions containing the first of the eluted material. At least 2 or 3 zones of activity observed. Activity associated with strong ultra-violet absorption at 260 m except for that found in the earliest zone eluted from column. Activity appears not to be associated with zones which give a strong ninhydrin reaction.

Crystalline Compounds "13 and "C" isolated. Crystalline phenylalanine isolated (inactive).

Remainder of crude O. M. F. concentrate No. 1 equivalent to 350 gallons of coconut milk solvent-fractionated preliminary to chromatographic separation. Some degree of activity found in all fractions, roughly proportional to intensity of ultraviolet absorption. Free. 1. 14 gms. insol. ethanol, u. v. extinct. coefl.=2.l Approx. 10 gms. of inorganic salts Free. 2. 220 gms. insol. ethanol, 1:. v. extinct. coefl.=5.9

Free. 3. 35 gms. imol. ethanol, 11. v. extinct. coefi.=7.35

Frac. 4. 27 gms. insol. absol. ethanol, u. v. extinct. coefi.=10.0 irorn 1.0 gm. of fraction 53 mg. pure alanine obtained (inactive) V V Frac. 5. 2.9 crude solid separating from cold absol. ethanol, u. v. extinct. coeft.=7.3 0.8 gm. crystalline pipecolic acid hydrochloride (inactive) Frac. 6. 25 gms. syrup separating irom chilled absol. ethanol on longer storage. from 2.0 gm. of traction mg. crude leucine obtained (inactive) u. v. extinct. coefi.=14.9

Frac. 7. 19 gms. insol. in 50/50 ethanol-acetone, u. v. extinct. coefi.=30.2 6.0 gms. chromatographed on cellulose Free. 8. 33 gms. insol. in 10/90 ethanol-benzene, u. v. extinct. coefl.=6.0

Free. 9. 2.6 gms. soluble in benzene, u. v. extinct coeff.=21.5

' Isolation of Compound F Crude C. M. F. concentrate No. 1, passedthrusteps designatedFractions6and7asshownab0ve. Fractions 6 and 7 combined and, except for the 6 grams chromatographed as shown above the combined residue treated as iollows:

Mixed with cellulose powder and dried. Dry material packed in a large chromatography column.

Washed with solvents in graded polarity series from other, fcelilzonehausilute alcohol, aqueous alcohol and ending with water. 90% a co 0 a e 90% ethanol eluate Ohromatographed on Amberiite XE64 (a mild carboxylic acid resin), developed and washed with distilled water to remove some fractions. Bands of ultraviolet absorbing material, one of which was Compound B, were removed.

Washed with 0.1 N hydrochloric acid. First material was a fairly sharp hand with strong ultraviolet band at 219 and 269 my in acid, at 224 and 280 my in base, and an extinction coeflicient at 269 1111.: in acid of 41.

Product with extinction coefficient at 269 mu= 162.

Dlssolved'ln-hot .watenandcooled. A- whlte non-crystallinepowder appears T at 269"m;r=

Extinction eoefiicient It should be noted that the substances obtairiedfrom coconut milk by the processes of this invention, designated for convenience as Compounds A, B, C and F, fail to produce at any concentration a total response which approaches that given by the addition of whole coconut milk.

To our surprise, We found that the full response could be restored by adding an enzymatic hydrolysate of casein (for which the coconut milk is a non-specific source) to the basal medium at a level of 500 p. p. m., and to a somewhat lesser extent by the addition of pure amino acid 5 mixtures.

The addition .of casein 'hydrolysatetalone; to the basal. medium has but a relatively slight effect upon growth; a pronounced response is obtained only in combination with certain fractions from coconut milk.

Following our: discovery. of the-effect ofJadded" casein: hydrolysate, various fractions of the coconut milk concentrate have been examined to determine which ones, in-

effective in themselves, are active when tested in the presence'mf casein hydrolysate. This has-been done by measuring the growth of aseptic carrot tissue eXplants in an otherwise synthetic medium. From fractions which proved to be active in this assay procedure we have isolated four substances, referred to hereafter as Compounds A, B, C and P, which, when tested in the presence of casein hydrolysate, induce a rate of growth which approaches that obtained by the use of whole coconut milk. These substances discussed more fully hereafter in an individual fashion are all characterized by being ninhydrin inactive. It is believed that some of them contain their nitrogen in the pyridine nucleus.

Compound A.(J,3-diphenylurea) This crystalline substance was obtained directly upon, evaporation of the alcohol extracttdescribed above. The alcohol-soluble portion. ofithe mercury-freed precipitate from 800 gallons of coconut milk was reduced to three liters of aqueous solution. This was filtered to remove a small amount of insoluble residue. Upon drying the filter paper a numben of sm allwhite crystals .were; observed and mechanically separated from extraneous material. The 78 mg. of crude crystals thus obtained were twice recrystallized from 2 ml. of hot absolute ethanol, giving a final yield-of56 mgr ,ofifineiwhite needlesimelting at 240.5 C. (uncor) The maximum solubility of compound A in water at room temperature is about 40 mg. per liter. The ultraviolet absorption curve in absolute ethanol is shown in 7 Figure l. E (1%, 1 cm.) at the maximum (256 my): 1710. The infrared absorption curve of compound A in a Nujol mull is shown in Figure 2. The strong absorption bands at 3.45 and 7.26 p and part of the band at 6.90 a are due'to th'eiNujoli. COI'IIPOUIIGI'A givesno color reaction with ninhydrin. Anal. C, 73.58; H, 5.78; V N, 13.17;'O, 7.63. Molecular weight as determined by isothermal distillation l73230.

The growth response-ofCompound A aswwellas of Compounds B and C in the c'arrot'issue bioassay test-as: described by S. M. Caplin and F. C. Steward in Nature 163, 920 (1949), is shown graphically in Figure 6. Each 75 Peak fractions taken, alcohol to give an extinction coelficient at 269 m equal to 193.

established by paper chromatogr were obtained after filtering,

concentrated in aqueous acid, precipitated by absolute agld) is collected. This powderis- Compound F? bar in Figure 6"repre'se nts the average weight of'at least 3 carrot phloem explants after a l4-day test period. The original weight of each explantgequals 2.6 mg. These tests were not done concurrently and therefore each of the three gronpscdepicts the;growth-20fv the-explant: from:- a difierent carrot root.

TABLE 1 I oontronnn B nroassar Final Average Fresh N0. of

Weight, Cultures Basal 4. lit). 4 4 Basal+0oconut Milk 53. 1=|:12. 2 4 Basal-l-Caseln Hydrolysate 12. 0:);2. 7 4 Basal+Compound B, 0.25 p., p. m 7. 8=i=4. 4 4.. Basal+0asein HYdrolysate-i- Compound-'13.. 50.'8i7. 7 3

COMPOUND O BIOASSAY Basal V 4 Basal-l-Coconut Milk 4 Basal-I-Casein Hydrolysate 4 Basal+0ompound G, 2.0 p. p. m 4 Basal+0asein Hydro1ysate+Gompound O 4 A portion of 'the original crude concentrate equiv-' alent to about 200 gallons of the original coconut milk was further enriched by solvent fractionation to yield 2.8 g. of material active in the growth assay at 20 p. p. m. This concentrate was chromatographed" in n-butanolacetic acid-water mixture on a column containing 800 g. of finely powdered cellulose and was divided into 300 fractions of,25.ml. each. Each fraction was examined for its ultraviolet'absorption, fluorescence under u1tra-' violet, and. intensity of its reaction, if any, with ninhydrin.

About;5.0.% of.-:the:materialpreceded the first strong ninhydrin-reaction region. Strong biological'activity was found distributed throughout these early fractions, and further fractionation of material from this region indicates that .there: are; several: active substances, identifiable by differences in ultraviolet absorption or, in at least one instance, by an intense blue fluorescence.

From the fastest moving ninhydrin-reaction region crystalline. phenylalanine Jwas; obtained and its identity aphy. From a later region of fractions, showing both a strong ninhydrin test and ultraviolet absorption at 275 m crystalline tyrosine was obtained. Between the phenylalanine and tyrosine regions there were fractions which absorbed strongly at 260-275 m but did not react with ninhydrin. From these fractions Compounds B and C were isolated.

Compound B.

Crystalline Compound B was obtained from the fractions immediately preceding the tyrosine upon evaporating to a small volume. The crystals were filtered, collected,- washedwithalcohol, redissolved in water, and ultimatelyrecrystallized'from 0.3 ml. of absolute alcohol. One and two-tenth milligrams of small white needles washing and drying. From red region of the spectrum the adjoining fractions an additional 0.7 mg. was'ob-' tained. This compound has a distinctive ultraviolet absorption curve with a pronounced shift between acid and alkaline solution.

In Figure 3 there is shown the ultraviolet absorption curve of Compound B in 0.001 N HCl (solid line) and .in 0.001 N NaOH solution (dotted line). The values of E (1%, 1 cm.) at the maxima are 568 at 267 m in acid solution, and 470 at 240 my. and 475 at 277 m in alkaline solution.

Compound B exhibits absorption bands in the infra- (determined in the solid state) at the following wavelengths and relative intensities:

7 Compound F This powdery substance has been obtained by the cel-' lulose column partition chromatography procedure outlined above. It is active in the carrot assay in a manner similar to Compounds A, B and C. The compound has a distinctive ultraviolet absorption curve. The value of E (1%, 1 cm.) at the maxima 1540 at 219 1111.; in acid solution; and 280 at 280 my. and 1360 at 224 my in alkaline solution.

Compound F exhibits absorption bands in the infrared region of the spectrum (determined in the solid state using Relative band A Rqlative band the lsiBndisc technique) at the following wavelengths and mtenslty intensity relative mtensmes:

2. strong. medium.

- A Relative band 7\ Relative band 3. medium. weak. 3. strong, broad. Do. intensity Intensity 15. 0. b d medliDum me um, roa 0. 4. weak, broad. D0. 3%: fia 2:22 zfg l g. veryDvgeak. Ve g 3.62- weak, 8.73 D0. e g, 3.80- very weak. 8.99 D0.

Tong 4.07. D0. 9.30 medium, broad.

2 i 323 broad 6.11- very strong. 9.86 Do. a 5 5 6.72 very weak. 10.0 weak. me ak 6.78 Do. 10.5 weak, broad.

------------ we 6.88- medium. 11.19 Do.

7. strong. 11. D0. 7.28.-. weak. 1;. Compound C 3133::

This crystalline substance has been obtained from the 31%;: g

fractions immediately following phenylalanine in the cellulose column partition chromatography procedure. These fractions (210 mg.) were dissolved in 50 m1. of hot acetone, the volume reduced to 10 m1. and solution stored for a period of two days at 20 C. Clusters of prismatic white crystals were obtained which were filtered, washed with cold acetone, recrystallized twice from hot acetone, washed and dried to give a yield of about 20 mg.

When determining the melting point it was observed that the material sublimed at ZOO-210 C. and condensed in a crystalline state on the cool portions of the capillary. By ultraviolet and infrared absorption, the sublimed material was found to be apparently unchanged and its biological activity in the growth test also with stood this procedure.

The ultraviolet absorption curve of Compound C in acid (0.001 N HCl-solid line) and alkaline solution (0.001 N NaOH) is shown in Figure 4. In alkaline solution the absorption maximum at 263 m is markedly depressed but unchanged in position. The values of E (1%, 1 cm.) at the maximum (263 my) are 326 in acid solution and 177 in alkaline solution; and at the minimum (232 my) the values of E are 31 in acid solution and 51 in alkaline solution.

The infrared absorption spectrum of Compound C when dissolved in Nujol is shown in Figure 5. Wavelength values for the absorption bands and their relative intensities are as follows:

7t Relative band A Relative band intensity intensity strong. 8.10. medium. medium. 8.70- D0. medium, broad. 8.95- weak.

Do. 9.05 very weak. medium. 9.40. medium.

D0. 9.05. Do.

. strong. 10.15 Do. medium. 10.45 weak. weak. 10.75-. D0. medium. 10.95 Do.

weak. 11.05

medium. 12.02 very weak. 13.10 medium. 13.95

Compound F has been found to have growth-promoting activity on carrot explants from certain stocks.

These novel growth-promoting substances particularly when used in combination with casein hydrolysate are of use as synergists for 2,4-D in herbicidal compositions as described and claimed in the copending United States application of Frederick C. Steward and Samuel M. Caplin, Serial No. 473,006, December 3, 1954, which is a continuation-in-part of Serial No. 285,411, filed May 1, 1952, now abandoned.

The compounds of our invention are also useful when admixed with a nutrient medium containing certain plant extracts to inhibit the growth of plant tissues as fully described and claimed in the copending United States applications of Frederick C. Steward and Samuel M. Caplin, Serial Nos. 473,007 and 473,008, filed December 3, 1954, both of which are continuations-in-part of Serial No. 285,412, filed May 1, 1952, now abandoned.

We claim:

1. In a process for obtaining from coconut milk chemical substances capable of fostering in the presence of a basal medium containing casein hydrolysate rapid and random division of cells of higher plants, the steps which comprise diluting whole coconut milk with alcohol, treating the alcoholic solution with an excess of mercuric acetate, filtering the reaction mixture, collecting the resulting precipitate, suspending the precipitate in water, treating said precipitate with hydrogen sulfide, filtering off the precipitated sulfide, concentrating the filtrate to a heavy sludge, and extracting said sludge with an alcohol.

2. In a process for obtaining from coconut milk chemical substances capable of fostering in the presence of a basal medium containing casein hydrolysate rapid and random division of cells of higher plants, the steps which comprise mixing coconut milk at about pH 5.0 with an equal volume of ethanol and an excess of mercuric acetate, filtering the mixture, suspending in water the precipitate obtained upon filtration, treating the suspended precipitate with hydrogen sulfide, removing the precipitated sulfide by filtration, collecting the filtrate, concentrating the filtrate to a sludge under reduced pressure and extracting the sludge by agitation with ethanol.

are 310 at 269 my. and" 3;? In, aprocess :for obtaining-from V coconutemilk chemicalvsubstances capable-of ifosteringiin the; presence, of;a,. basal; medium containing casein; hydrolysate; rapid; and:

random division of cells of higher plants, the steps which comprise mixing coconut at about pH 5.0 with an equal volumeof ethanol and an'excessioflmercuric,acetate,

filtering the mixture,,.suspending;-in water: the precipitate? upon filtration treatingthe suspended precipitate; with; hydrogen sulfide, removing theprecipitated sulfide: by; filtration, collecting thefiltrate, concentrating the; 111- obtained irate to a tsludge underreducedpressure, extractingthe sludge by agitation :With190 extractby partition chromatography on ,QCIIUIOSEL.

; .lngthe processtof claim,3-, the step,ofifiactionating the product by partition chromatography on 'cellulose.

being effected in the presence of n-butyl alcohol saturated with 2M acetic acid.

5,. Achemical compound capable of ,fostering rapid and random division of mature'cells of higher plants ethanol, removing;at least a portion of the ethanohandfractionating the-resulting;

unchanged at 200*210 andVF; said. Compound B, being slightly. soluble in a1cohol and-,havingultraviolet absorption spectrum maxima: at. 267 mp -in.acid solution, B (1%, 1 cm.)=568,: and at 240 my and 277 m in alkaline solution, B (1%,

. 1 cm.) ,=470,'- and-475; and an infrared-spectrum showing strong bands; at the following cwavelengths expressed ino 3.49;,(broad), and 5.84; and;saidComp oundjC being soluble in hot acetone but;

microns: 2.92, 3.221: (broad),

insoluble in cold acetone, andhavingrultraviolet absorption'spectrum at 263 my in acid,.E'(1%,' and in alkaline solution,- E (1%, 1 cm;)=177, exhibiting strong bandstin'can infrared absorptionspectrumatth: following=wavelengthso expressed in microns: 2.95, 6.05, 1 and 12.02, and being further characterized by subliming C. said Compound Ft having ultrat violet absorption spectra maximaoat 269v m and 219 m in acid, E; (1%, 1 cm.)=310 and-219', and at 280 m and 224mn'in alkaline-solutiom E- (1%, 1-cm:)=280" and 136.0, andt'an infrared spectrum showingstrongbands at the following wavelengths expressed inmicrons: 229 4; 7

' 3.18, 6.11, 7.13, and 13.60.

. No references cited;

1 cm.) =326, i 

1. IN A PROCESS FOR OBTAINING FROM COCONUT MILK CHEMICAL SUBSTANCES CAPABLE OF FOSTERING IN THE PRESENCE BASAL MEDIUM CONTAINING CASEIN HYDROLYSATE RAPID AND RANDOM DIVISION OF CELLS OF HIGHER PLANTS, THE STEPS WHICH COMPRISE DILUTING WHOLE COCONUT MILK WITH ALCOHOL, TREATING THE ALCOHOL SOLUTION WITH AN EXCESS OF MERCURIC ACETATE, FILTERING THE REACTION MIXTURE, COLLECTING THE RESULTING PRECIPITATE WITH HYDROGEN SULFIDE, FILTERING TREATING SAID PRECIPITATE WITH HYDROGEN SULFIDE, FILTERING OFF THE PRECIPITATED SULFIDE, CONCENTRATING THE FILTRATE HEAVY SLUDGE, AND EXTRACTING SAID SLUDGE WITH AN ALCOHOL. 